Severe Dopaminergic Neurotoxicity in Primates After a Common Recreational Dose Regimen of MDMA ("Ecstasy")

The prevailing view is that the popular recreational drug (±)3,4-methylenedioxymethamphetamine (MDMA, or "ecstasy") is a selectiveserotonin neurotoxin in animals and possibly in humans. Nonhumanprimates exposed to several sequential doses of MDMA, a regimenmodeled after one used by humans, developed severe brain dopaminergicneurotoxicity, in addition to less pronounced serotonergic neurotoxicity.MDMA neurotoxicity was associated with increased vulnerabilityto motor dysfunction secondary to dopamine depletion. These resultshave implications for mechanisms of MDMA neurotoxicity and suggestthat recreational MDMA users may unwittingly be putting themselvesat risk, either as young adults or later in life, for developingneuropsychiatric disorders related to brain dopamine and/or serotonindeficiency.

MDMA ("ecstasy") has become a popular recreational drug internationally (1, 2). In the 1980s, MDMA was generallyused on college campuses, with most individuals taking no morethan one or two 75- to 150-mg doses, about 1.6 to 2.4 mg per kilogramof body weight (mg/kg), twice monthly (3). More recently,MDMA is increasingly used in the context of large, all-night danceparties where partygoers regard the drug as safe and consume multipledoses during the night (4, 5).

MDMA appears to carry risks beyond the sociobehavioral effects associated with drug abuse. Experimental animals treated withMDMA show evidence of brain serotonin neurotoxicity (6-8),and MDMA-induced serotonin neurotoxicity may also occur in humans(9, 10). Virtually all animal species testeduntil now show long-term effects on brain serotonin neurons butno lasting effects on either brain dopamine or norepinephrine(NE) neurons (6-8). In the mouse, dopamineneurons are affect ed, but serotonin neurons are spared (11,12).

We used nonhuman primates to evaluate the neurotoxic potential of a dose regimen modeled closely after one often used by MDMAusers at all-night dance parties. Squirrel monkeys (Saimiri sciureus)were given MDMA at a dosage of 2 mg/kg, three times, at 3-hourintervals, for a total dose of 6 mg/kg (13). Of fivemonkeys treated with MDMA, three tolerated drug treatment withoutany apparent difficulty. One monkey became less mobile and hadan unstable, tentative gait after the second dose, and thereforeit was not given the third planned dose. The fifth monkey developedmalignant hyperthermia and died within hours of receiving thelast dose of MDMA. Two weeks after MDMA treatment, the three monkeysthat tolerated drug treatment were examined for chemical and anatomicmarkers of brain serotonin neurons (13), along withthree saline-treated control animals. These studies revealed lastingreductions in regional brain serotonin, serotonin's major metabolite(5-hydroxyindoleacetic acid, or 5-HIAA), and the serotonin transporter(SERT). Anatomic studies (13) supported these observations,showing reductions in the density of serotonin- and SERT-immunoreactive(SERT-IR) axons in some cortical regions (Fig. 1). Sixweeks after MDMA treatment, the monkey that received only twodoses of MDMA was evaluated and found to also have long-lastingreductions in serotonin axonal markers; serotonin, 5-HIAA, andSERT in the caudate nucleus of this animal were reduced by 37,48, and 40%, respectively.

These same monkeys had marked reductions in various markers of striatal dopaminergic axons (Fig. 2). The profoundloss of striatal dopaminergic axonal markers was consistentlyobserved in all monkeys examined, including the animal that receivedonly two MDMA doses; dopamine, 3,4-dihydroxyphenylacetic acid(DOPAC), and the dopamine transporter (DAT) in the caudate nucleusof this animal were reduced by 65, 77, and 51%, respectively,6 weeks after MDMA exposure. The loss of dopaminergic axonal markerswas greater than the loss of serotonergic axonal markers. Morphologicstudies revealed corresponding reductions in the density of striatalDAT- and tyrosine hydroxylase (TH)-IR axons throughout the striatalcomplex, with some sparing of the more caudal portion of the caudatenucleus (Fig. 2). Quantitative autoradiography studies(13) confirmed the severe reductions in striatal DATdensity (Fig. 2).

Fig. 2. Effect of MDMA treatment on striatal concentrations of (A) dopamine (DA), (B) [3H]WIN35,428-labeled DAT, (C) DOPAC, and (D) radioisotope [3H]MTBZ-labeled vesicular monoamine transporter-2 (VMAT) in squirrel monkeys examined 2 weeks after MDMA treatment. (E) [3H]RTI-121-labeled DAT in coronal section of a control monkey and a monkey treated with MDMA 2 weeks previously. The scale on the right shows the density of binding sites designated by color expressed in nCi/mg of tissue. (F and G) DAT-IR axons and axon terminals in the striatum of (F) a control monkey and (G) a monkey treated with MDMA 2 weeks previously. (H and I) TH-IR axons and axon terminals in the striatum of (H) a control monkey and (I) a monkey treated with MDMA 2 weeks previously. Dark-field photomicrographs of the sagittal plane are shown; scale bar = 100 µm.

To determine whether the severe long-lasting decrements in dopaminergic axonal markers in squirrel monkeys were unique tothis primate species, we tested the effects of the same MDMA regimenin baboons (Papio anubis) (13). Again, one of five animalsdied, this time shortly after receiving only two doses of MDMA.Malignant hyperthermia (up to 41.6oC) was again an important factor. A second baboon appeared unstableafter the second dose of MDMA and therefore received only twoof the three planned doses. Two to 8 weeks after treatment, thefour surviving MDMA-treated baboons, along with three saline-treatedcontrol animals, underwent chemical and anatomic studies of braindopamine and serotonin neurons (13). Neurochemical andquantitative autoradiography studies again revealed a profoundloss of striatal dopaminergic axonal markers (Fig. 3).Dopaminergic deficits in the striatum of the baboon that receivedonly two MDMA doses were as severe as those in the baboons thatreceived all three doses. Baboons also developed less severe,but significant, long-term reductions in regional brain serotonergicneuronal markers (Fig. 3).

Fig. 3. Effect of MDMA treatment on striatal concentrations of (A) dopamine, (B) [3H]WIN35,428-labeled DAT, (C) DOPAC, and (D) [3H]MTBZ-labeled VMAT in baboons examined 2 weeks after MDMA treatment. (E) [3H]RTI-121-labeled DAT in a coronal section of a control baboon and a baboon treated with MDMA 2 weeks previously. The scale on the right shows the density of binding sites designated by color expressed in nCi/mg of tissue. (F) Serotonin (5-HT), (G) 5-HIAA, and (H) SERT in baboons 2 weeks after MDMA treatment. (I) [3H]RTI-55-labeled SERT in a coronal section of a control baboon and a baboon treated with MDMA 2 weeks previously. The scale on the right shows the density of binding sites designated by color expressed in nCi/mg of tissue.

To evaluate the selectivity of the observed effects, we assessed the status of noradrenergic neurons in both monkeys and baboons.MDMA produced no long-term effects on NE levels or the densityof NE transporters in the brain of either primate species (figs.S1 and S2). Consistent with the lack of a long-term effect ofMDMA on the concentrations of NE and its transporter, the densityof TH-IR axons in the cerebral cortex of MDMA-treated monkeyswas unaffected (fig. S1).

To determine that the lasting loss of chemical and anatomic markers of striatal dopaminergic and serotonergic axons and axonterminals was, in fact, due to a neurotoxic insult rather thanto lingering acute pharmacological effects of MDMA, we used Finkand Heimer's method (14), which allows for selectivesilver impregnation of degenerating axons and axon terminals.A monkey treated with MDMA and evaluated 31/2 days later (13)had dense argyrophilic debris characteristic of axon terminaldegeneration in the striatum (Fig. 4). No such degenerativedebris was evident in the striatum of the control animal. We alsofound a vigorous glial response (Fig. 4) in adjacentstriatal tissue sections processed for glial fibrillary acidicprotein (GFAP) immunocytochemistry (13).

Fig. 4. Silver-stained coronal sections through the caudate nucleus of (A) a control monkey and (B) a monkey treated with MDMA (one dose of 2 mg/kg at 3-hour intervals, three times) 31/2 days previously. Fine argyrophilic debris in the MDMA-treated monkey is characteristic of axon terminal degeneration, as demonstrated by the Fink-Heimer method (14). Scale bar = 10 µm. (C) Paucity of GFAP-IR cells in the caudate nucleus of a control monkey and (D) marked increase in the number of GFAP-IR cells in the striatum of a monkey treated with MDMA 31/2 days previously. Scale bar = 10 µm.

We next explored the possibility that monkeys with MDMA-induced dopaminergic neurotoxicity (with no evidence of Parkinsonism)are at increased risk for the development of motor dysfunctionsecondary to dopamine depletion (13). Monkeys (n = 3)received a challenge dose regimen of alpha-methyl-para-tyrosine(AMPT) 1 week before and 1 week after MDMA treatment. Using adosage regimen of AMPT that gradually reduces brain dopamine concentrations,we hoped to model the progressive decline in brain dopaminergicfunction that occurs with normal aging (15). Comparedto their baseline, monkeys were more sensitive to AMPT-inducedmotor dysfunction 1 week after MDMA treatment (fig. S3).

We report severe, functionally significant dopaminergic neurotoxicity, along with more modest serotonergic neurotoxicity,in primates treated with doses of MDMA modeled after those commonlyused by recreational MDMA users. Earlier studies in nonhuman primateshave generally involved administration of higher MDMA doses (5or 10 mg/kg) twice daily (morning and evening) for 4 consecutivedays. These dosage regimens typically engendered more severe buthighly selective toxicity toward brain serotonin neurons, withno long-term effects on brain dopamine neurons (16-18).Because the drug regimens used in previous studies did not modelthose used by most MDMA users, the possibility remained that occasionalMDMA users might not be at risk for neurotoxic injury. The presentresults, however, indicate that even individuals who use MDMAon one occasion may be at risk for substantial brain injury ifthey use two or three sequential doses, hours apart, as is oftenthe case in recreational settings.

In the present studies, MDMA was given by a systemic route (subcutaneously in squirrel monkeys and intramuscularly in baboons),whereas humans generally take MDMA orally. It is possible thathumans are at a decreased risk for neurotoxic injury because ofdifferences in the route of administration. However, in the caseof MDMA, oral administration offers little or no significant neuroprotection(19-22). Even if some degree of protectionwere afforded by oral administration, the profound loss of dopaminergicneuronal markers seen in both primate species suggests that significantneurotoxicity would still occur. Moreover, individual doses ofMDMA used in this study are lower than those typically used byhumans (1.6 to 2.4 mg/kg), once adjusted with interspecies dosescaling methods (23). Hence, any protection that mightbe associated with oral administration would likely be offsetby increasing the dose of MDMA used in this study to the humanequivalent. It is not uncommon for recreational MDMA users touse repeated doses of the drug on more than one occasion or morethan two or three repeated doses per session.

The present findings challenge the commonly held notion that MDMA is a selective brain serotonin neurotoxin and carry importantpublic health and scientific implications. Based on MDMA use pattern,there may be two separate MDMA cohorts: those with selective brainserotonergic neurotoxicity and those with combined serotonergicand more severe dopaminergic neural injury. It will be exceedinglyimportant to consider this when attempting to identify and interpretfunctional consequences of MDMA use in humans. Cognitive abnormalitiesidentified in MDMA users (24-26) may berelated, at least in part, to dopaminergic rather than serotonergicneurotoxicity. The present findings also have implications forefforts aimed at identifying the mechanisms of MDMA neurotoxicity.Previous studies have identified a metabolite of MDMA that mightbe responsible for its neurotoxic effects, the 6-hydroxydopamineanalog 2-(methylamino)-1-(2,4,5-trihydroxyphenyl) propane (27-29).Because this toxic metabolite induced both dopaminergic and serotonergicneurotoxicity, and because MDMA was believed to be a selectiveserotonin neurotoxin, it received little further attention. This6-hydroxydopamine analog of MDMA obviously warrants closer scrutinyas a potential mediator of MDMA neurotoxicity.

The development of profound dopaminergic neurotoxicity after two or three sequential MDMA doses of 2 mg/kg each leads oneto question what distinguishes this particular drug regimen fromthe 4-day, twice daily, higher-dose regimen that engenders selectiveserotonergic neurotoxicity (16-22).One possibility is that the nonlinear pharmacokinetic profileof MDMA, such as that demonstrated in humans in the setting ofclosely spaced repeated dosing (30, 31), leadsto prolonged elevated brain levels of MDMA (or its metabolites)and that protracted exposure to MDMA renders dopamine neuronsvulnerable to its toxic effects. An alternative (although notmutually exclusive) explanation is that repeated closely spaceddoses of MDMA lead to higher elevations in body temperature, whichis known to augment MDMA neurotoxicity (32). Additionalstudies are needed to evaluate these possibilities, in additionto alternative hypotheses.

In light of the present findings, and given the fact that MDMA use is widespread and increasing, one might ask why more casesof MDMA-induced Parkinsonism (33) have not been reported.There are multiple potential explanations, but only two will bementioned. First, Parkinsonism does not generally become clinicallyapparent until more than 70 to 80% of brain dopamine has beendepleted. Therefore, substantial MDMA-induced dopaminergic neurotoxicitycould occur yet remain occult until unmasked by other processes(such as drug-induced interference with dopaminergic neurotransmissionor decline in brain dopamine with advancing age). Second, untilnow, the potential for MDMA to damage brain dopamine neurons inprimates has not been appreciated and, therefore, MDMA neurotoxicityhas not been considered in the differential diagnosis of Parkinsonismin young adults. It is possible that some of the more recent casesof suspected young-onset Parkinson's disease might be relatedto MDMA exposure but that this link has not been recognized.

These findings suggest that humans who use repeated doses of MDMA over several hours are at high risk for incurring severebrain dopaminergic neural injury (along with significant serotonergicneurotoxicity). This injury, together with the decline in dopaminergicfunction known to occur with age (15), may put theseindividuals at increased risk for developing Parkinsonism andother neuropsychiatric diseases involving brain dopamine/serotonindeficiency, either as young adults or later in life.